Engineered red blood cell-based biosensors

ABSTRACT

Disclosed are systems and methods for detecting extracellular ligands. The disclosed systems and method for detecting extracellular ligands typically comprise or utilize engineered red blood cells (eRBCs) that comprises modular extracellular sensors. The eRBCs may comprise: (i) a first exogenous extracellular sensor; the first extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a first fragment of a functional protein, and (ii) a second exogenous extracellular sensor; the second extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a second fragment of the functional protein. In the eRBCs, the ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand to form a tertiary complex, and the first fragment of the functional protein and the second fragment of the functional protein interact in the tertiary complex to reconstitute functional activity of the functional protein. Suitable functional proteins for the disclosed eRBCs may include fluorescent proteins that emit fluorescence when the ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand, and enzymatic proteins that exhibit enzymatic activity when ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under11-23-CCM-DT-FP-008 awarded by The Defense Advanced Research ProjectsAgency (DARPA). The government has certain rights in the invention

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.15/908,077, filed Feb. 28, 2018, which claims the benefit of priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/464,754,filed on Feb. 28, 2017, the content of which is incorporated herein byreference in its entirety.

BACKGROUND

The present invention provides modular extracellular sensors, nucleicacids encoding such sensors, cells expressing such sensors, systems andmethods of employing such sensors and cells for detecting extracellularligands. In particular, the invention relates to red blood cells thathave been engineered to express modular extracellular sensors fordetecting extracellular ligands.

SUMMARY

Disclosed are systems and methods for detecting extracellular ligands.The disclosed systems and method for detecting extracellular ligandstypically comprise or utilize engineered red blood cells (eRBCs) thatcomprises modular extracellular sensors. The eRBCs may comprise: (i) afirst exogenous extracellular sensor; the first extracellular sensorcomprising: a) an extracellular ligand binding domain or a portionthereof, b) a transmembrane domain, and c) a first fragment of afunctional protein, and (ii) a second exogenous extracellular sensor;the second extracellular sensor comprising: a) an extracellular ligandbinding domain or a portion thereof, b) a transmembrane domain, and c) asecond fragment of the functional protein. In the eRBCs, theextracellular ligand binding domain of the first exogenous sensor andthe ligand binding domain of the second exogenous sensor bind to aligand to form a ternary complex (which may be the same ligand), and thefirst fragment of the functional protein and the second fragment of thefunctional protein interact in the ternary complex to reconstitutefunctional activity of the functional protein.

The first exogenous sensor and/or the second exogenous sensor may bederived, prepared, and/or engineered from native membrane proteins ofRBCs or portions or fragments thereof. For example, the first exogenoussensor and/or the second exogenous sensor may be derived, prepared,and/or engineered from a portion of a native membrane protein of a RBCcomprising at least the transmembrane portion to which is fused a ligandbinding domain at the extracellular terminus and a functional protein(or a portion of a functional protein) at the intracellular terminus.The transmembrane portion further may be mutated in order to preventhomodimerization of the transmembrane portion with another transmembraneportion, for example, where the native membrane protein form a homodimerin native conditions.

Suitable functional proteins for the disclosed eRBCs may includefluorescent proteins that emit fluorescence when the ligand bindingdomain of the first exogenous sensor and the ligand binding domain ofthe second exogenous sensor bind to the same ligand, and enzymaticproteins that exhibit enzymatic activity when ligand binding domain ofthe first exogenous sensor and the ligand binding domain of the secondexogenous sensor bind to the same ligand. Substrates for the enzymaticproteins may include substrates that are luminescent after they aremetabolized by the enzymatic protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Proposed mechanism of protein-based biosensors.

FIG. 2: Evaluation of first-generation biosensor proteins containing asimple transmembrane domain. All samples were analyzed in biologicaltriplicate, the mean was subtracted from the mean of negative controlcells (expressing only one receptor chain), and error bars represent onestandard deviation. Abbreviations: RBD: Rapamycin Binding Domain—FRB orFKBP; sGFP: split GFP—GS (small fragment) or GL (large fragment)

FIG. 3: Proposed mechanism of Kell-based biosensor protein and GPA-basedbiosensor protein. Kell and GPA, two native RBC proteins, were modifiedto contain extracellular rapamycin-binding domains and intracellularsplit GFP halves.

FIG. 4: Evaluation of Kell-based biosensor protein and GPA-basedbiosensor protein. All samples were analyzed in biological triplicate,the mean was subtracted from the mean of cells transfected with only onereceptor half, and error bars represent one standard deviation.Abbreviations: RBD: Rapamycin Binding Domain—FRB or FKBP; sGFP: splitGFP—GS (small fragment) or GL (large fragment); RBC: Red Blood CellProtein.

FIG. 5: Proposed mechanism of GPA only-based biosensor proteins.

FIG. 6: Evaluation of GPA only-based biosensor proteins compared toGPA-based and Kell-based biosensor proteins. All samples were analyzedin biological triplicate, the background auto-fluorescence of the cellswith only the color control was subtracted off, and error bars representone standard deviation. Abbreviations: Rap: Rapamycin Binding Domain—FRBor FKBP; GFP Half: split GFP—GS (small fragment) or GL (large fragment);RBC: Red Blood Cell Protein.

FIG. 7: Second generation proposed mechanism of mutated GPA (GPAm)-basedbiosensor proteins. Abbreviations: Rap: Rapamycin Binding Domain—FRB orFKBP; GFP Half: split GFP—GS (small fragment) or GL (large fragment);RBC: Red Blood Cell Protein.

FIG. 8: Mutation-driven improvement of eRBC biosensors. All samples wereanalyzed in biological triplicate, the mean was subtracted from the meanof negative control cells, and error bars represent one standarddeviation. Abbreviations: FD: fold-difference; GPA Mutant: WT: WildType, V84R: Valine 84 to Arginine, V84E: Valine 84 to Glutamic Acid,V84K: Valine 84 to Lysine.

FIG. 9: Evaluation of mutated GPA-based biosensor proteins compared toGPA-based and Kell-based biosensor proteins. All samples were analyzedin biological triplicate, error bars represent one standard deviation.Abbreviations: Rap: Rapamycin Binding Domain—FRB or FKBP; GFP Half:split GFP—GS (small fragment) or GL (large fragment); RBC: Red BloodCell Protein; Mutate: X—wild type or V84R: Valine 84 to Argininemutation.

FIG. 10: Third generation proposed mechanism of mutated GPA-basedbiosensor proteins.

FIG. 11: Evaluation of GPA-based biosensor proteins compared to mutatedGPA-based biosensor proteins with NANOLUCIFERASE™ output. All sampleswere analyzed in biological triplicate, error bars represent onestandard deviation. Abbreviations: GPA mutant—X: wild type GPA, V84R:Valine 84 to Arginine; NANOLUCIFERASE™ Half—11S (larger half) or 114(smaller half); FD—fold difference.

FIG. 12: Evaluation of GPA-based biosensor proteins with NANOLUCIFERASE™output in G1er cells, which approximate red blood cells. Abbreviations:FD—fold difference.

FIG. 13: Evaluation of GPA-based biosensor proteins with NANOLUCIFERASE™output in human CD34+ cells.

DETAILED DESCRIPTION

The present invention is described herein using several definitions, asset forth below and throughout the application.

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a receptor,” “ligand,” and“complex” should be interpreted to mean “one or more receptors,”“ligands,” and “complexes,” respectively.

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of these terms which are not clear to persons ofordinary skill in the art given the context in which they are used,“about” and “approximately” will mean plus or minus?10% of theparticular term and “substantially” and “significantly” will mean plusor minus>10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising” in that these latterterms are “open” transitional terms that do not limit claims only to therecited elements succeeding these transitional terms. The term“consisting of,” while encompassed by the term “comprising,” should beinterpreted as a “closed” transitional term that limits claims only tothe recited elements succeeding this transitional term. The term“consisting essentially of,” while encompassed by the term “comprising,”should be interpreted as a “partially closed” transitional term whichpermits additional elements succeeding this transitional term, but onlyif those additional elements do not materially affect the basic andnovel characteristics of the claim.

The disclosed technology relates to “extracellular sensors.” Asdisclosed herein, an “extracellular sensor” is a molecule or a system ofmolecules that can be used to bind to a ligand and provide a detectableresponse based on binding the ligand. Extracellular sensors aredisclosed in the art. (See, e.g., Daringer et al., “ModularExtracellular Sensor Architecture for Engineering Mammalian Cell-basedDevices,” Nichole M. Daringer, Rachel M. Dudek, Kelly A. Schwarz, andJosh N. Leonard, ACS Synth. Biol. 2014, 3, 892-902, published Feb. 25,2014; WO 2013/022739, published on Feb. 14, 2013; and U.S. PublicationNo. 2014-0234851; the contents of which are incorporated herein byreference in their entireties).

The disclosed extracellular sensors typically include a ligand-bindingdomain of a ligand-binding protein. As contemplated herein, a“ligand-binding protein” is a macromolecule, typically a protein, whichbinds to a ligand. For example, a ligand-binding protein may include areceptor for a ligand or a portion of a receptor for a ligand, forexample, where the receptor is a membrane protein and the ligand-bindingprotein comprises the extracellular portion of the receptor that bindsan extracellular ligand. A suitable ligand for the ligand-bindingdomains of the disclosed extracellular sensors may include more than onebinding site for a ligand-binding protein. As such, a suitable ligandcan bind more than one ligand-binding domain of one or moreextracellular sensors as contemplated herein, and as such, a suitableligand may form a ternary or high order complex with two or moreextracellular sensors.

The disclosed exogenous sensors may be utilized for sensing anextracellular ligand and providing a molecular signal when the ligand issensed. Suitable molecular signals may be generated via so-called“bimolecular complementation” that occurs between two or more exogenoussensors (or between portions of two or more exogenous sensors) when theligand is sensed. “Bimolecular complementation” is known in the art, forexample, bimolecular fluorescence complementation and split enzymecomplementation or protein-fragment complementation are known in theart. (See Kodama et al., “Bimolecular fluorescence complementation(BiFC): A 5-year update and future perspectives, BioTechniques, Vol. 53,No. 5, November 2012, pp. 285-298. See also Remy et al., “Application ofprotein-fragment complementation assays in cell biology,” BioTechniques,Vol. 42, No. 2, 2007, pages 137-145; and Azad et al., “Split-luciferasecomplementary assay: Applications, recent developments, and futureperspectives,” Anal. and Bioanal. Chem 406(23) 2014, pages 5541-5560;the contents of which are incorporated herein by reference in theirentireties).

Reference is made herein to nucleic acid and nucleic acid sequences. Theterms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide,oligonucleotide, polynucleotide (which terms may be usedinterchangeably), or any fragment thereof. These phrases also refer toDNA or RNA of genomic or synthetic origin (which may be single-strandedor double-stranded and may represent the sense or the antisense strand).

Reference also is made herein to peptides, polypeptides, proteins andcompositions comprising peptides, polypeptides, and proteins. As usedherein, a polypeptide and/or protein is defined as a polymer of aminoacids, typically of length>100 amino acids (Garrett & Grisham,Biochemistry, 2nd edition, 1999, Brooks/Cole, 110). A peptide is definedas a short polymer of amino acids, of a length typically of 20 or lessamino acids, and more typically of a length of 12 or less amino acids(Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).

As disclosed herein, exemplary peptides, polypeptides, proteins maycomprise, consist essentially of, or consist of any reference amino acidsequence disclosed herein, or variants of the peptides, polypeptides,and proteins may comprise, consist essentially of, or consist of anamino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%,or 99% sequence identity to any amino acid sequence disclosed orcontemplated herein. Variant peptides, polypeptides, and proteins mayinclude peptides, polypeptides, and proteins having one or more aminoacid substitutions, deletions, additions and/or amino acid insertionsrelative to a reference peptide, polypeptide, or protein. Also disclosedare nucleic acid molecules that encode the disclosed peptides,polypeptides, and proteins (e.g., polynucleotides that encode any of thepeptides, polypeptides, and proteins disclosed herein and variantsthereof).

The term “amino acid,” includes but is not limited to amino acidscontained in the group consisting of alanine (Ala or A), cysteine (Cysor C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine(Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile orI), lysine (Lys or K), leucine (Leu or L), methionine (Met or M),asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q),arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine(Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. Theterm “amino acid residue” also may include amino acid residues containedin the group consisting of homocysteine, 2-Aminoadipic acid,N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, ?-alanine,?-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid,3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinicacid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid,allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine,3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid,6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine,Norvaline, 2,2′-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionicacid, Ornithine, and N-Ethylglycine. Typically, the amide linkages ofthe peptides are formed from an amino group of the backbone of one aminoacid and a carboxyl group of the backbone of another amino acid.

The amino acid sequences contemplated herein may include conservativeamino acid substitutions relative to a reference amino acid sequence.For example, a variant peptides, polypeptides, and proteins ascontemplated herein may include conservative amino acid substitutionsrelative to an amino acid sequence of a reference peptide, polypeptide,or protein. “Conservative amino acid substitutions” are thosesubstitutions that are predicted to interfere least with the propertiesof the reference peptide, polypeptide, or protein. In other words,conservative amino acid substitutions substantially conserve thestructure and the function of the reference peptide, polypeptide, orprotein. The following table provides a list of exemplary conservativeamino acid substitutions.

Table of Conservative Amino Acid Substitutions Original ResidueConservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, HisAsp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly AlaHis Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu MetLeu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe,Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

“Non-conservative amino acid substitutions” are those substitutions thatare predicted to interfere most with the properties of the referencepeptide, polypeptide, or protein. For example, a non-conservative aminoacid substitution might replace a basic amino acid at physiological pHsuch as Arg, His, or Lys, with a non-basic or acidic amino acid atphysiological pH such as Asp or Glu. A non-conservative amino acidsubstitution might replace a non-polar amino acid at physiological pHsuch as Ala, Gly, Ile, Leu, Phe, or Val, with a polar amino acid atphysiological pH such as Arg, Asp, Glu, His, or Lys.

Variants comprising deletions relative to a reference amino acidsequence or nucleotide sequence are contemplated herein. A “deletion”refers to a change in a reference amino acid sequence that results inthe absence of one or more amino acid residues. A deletion removes atleast 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or arange of amino acid residues bounded by any of these values (e.g., adeletion of 5-10 amino acids). A deletion may include an internaldeletion or a terminal deletion (e.g., an N-terminal truncation or aC-terminal truncation of a reference polypeptide). A “variant” of areference polypeptide sequence may include a deletion relative to thereference polypeptide sequence (e.g., relative to any of SEQ IDNOs:1-3).

The words “insertion” and “addition” refer to changes in an amino acidsequence resulting in the addition of one or more amino acid residues.An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, or 200 amino acid residues or a range of aminoacid residues bounded by any of these values (e.g., an insertion oraddition of 5-10 amino acids). A “variant” of a reference polypeptidesequence may include an insertion or addition relative to the referencepolypeptide sequence (e.g., relative to any of SEQ ID NOs:1-3).

A “fusion polypeptide” refers to a polypeptide comprising at theN-terminus, the C-terminus, or at both termini of its amino acidsequence a heterologous amino acid sequence, for example, a heterologousamino acid sequence that extends the half-life of the fusion polypeptidein serum. A “variant” of a reference polypeptide sequence may include afusion polypeptide comprising the reference polypeptide.

A “fragment” is a portion of an amino acid sequence which is identicalin sequence to but shorter in length than a reference sequence (e.g., afragment of any of SEQ ID NOs:1-3). A fragment may comprise up to theentire length of the reference sequence, minus at least one amino acidresidue. For example, a fragment may comprise from 5 to 1000 contiguousamino acid residues of a reference polypeptide. In some embodiments, afragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, 100, 150, 250, or 500 contiguous amino acid residues of areference polypeptide; or a fragment may comprise no more than 5, 10,15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguousamino acid residues of a reference polypeptide; or a fragment maycomprise a range of contiguous amino acid residues of a referencepolypeptide bounded by any of these values (e.g., 40-80 contiguous aminoacid residues). Fragments may be preferentially selected from certainregions of a molecule. The term “at least a fragment” encompasses thefull length polypeptide. A “variant” of a reference polypeptide sequencemay include a fragment of the reference polypeptide sequence.

“Homology” refers to sequence similarity or, interchangeably, sequenceidentity, between two or more polypeptide sequences. Homology, sequencesimilarity, and percentage sequence identity may be determined usingmethods in the art and described herein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide. Percent identity for amino acid sequences may bedetermined as understood in the art. (See, e.g., U.S. Pat. No.7,396,664, which is incorporated herein by reference in its entirety). Asuite of commonly used and freely available sequence comparisonalgorithms is provided by the National Center for BiotechnologyInformation (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul,S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available fromseveral sources, including the NCBI, Bethesda, Md., at its website. TheBLAST software suite includes various sequence analysis programsincluding “blastp,” that is used to align a known amino acid sequencewith other amino acids sequences from a variety of databases.

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 100, at least 150, at least 200, atleast 250, at least 300, at least 350, at least 400, at least 450, atleast 500, at least 550, at least 600, at least 650, or at least 700contiguous amino acid residues; or a fragment of no more than 15, 20,30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,or 700 amino acid residues; or over a range bounded by any of thesevalues (e.g., a range of 500-600 amino acid residues) Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in the tables, figures or SequenceListing, may be used to describe a length over which percentage identitymay be measured.

In some embodiments, a “variant” of a particular polypeptide sequencemay be defined as a polypeptide sequence having at least 20% sequenceidentity to the particular polypeptide sequence over a certain length ofone of the polypeptide sequences using blastp with the “BLAST 2Sequences” tool available at the National Center for BiotechnologyInformation's website. (See Tatiana A. Tatusova, Thomas L. Madden(1999), “Blast 2 sequences—a new tool for comparing protein andnucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such a pair ofpolypeptides may show, for example, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% orgreater sequence identity over a certain defined length of one of thepolypeptides, or range of percentage identity bounded by any of thesevalues (e.g., range of percentage identity of 80-99%).

The disclosed fusion polypeptides may comprise a amino acid sequencefused directly to a heterologous amino acid sequence or fused indirectlyvia a linker sequence. Suitable linker sequences may include amino acidsequences of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids ormore, or a range bounded by any of these values (e.g., a linker of 5-25amino acids). In some embodiments, the linker sequence comprises onlyglycine and serine residues.

Fusion polypeptide disclosed herein may include an amino acid tagsequence, for example, which may be utilized for purifying and oridentifying the fusion polypeptide. Suitable amino acid tag sequencesmay include, but are not limited to, histidine tag sequences comprising5-10 histidine residues.

A variant polypeptide may have substantially the same functionalactivity as a reference polypeptide. For example, a variant polypeptidemay exhibit or more biological activities associated with binding aligand, exhibiting fluorescence, and/or enzymatic activity.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences. Percent identity for anucleic acid sequence may be determined as understood in the art. (See,e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by referencein its entirety). A suite of commonly used and freely available sequencecomparison algorithms is provided by the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), whichis available from several sources, including the NCBI, Bethesda, Md., atits website. The BLAST software suite includes various sequence analysisprograms including “blastn,” that is used to align a knownpolynucleotide sequence with other polynucleotide sequences from avariety of databases. Also available is a tool called “BLAST 2Sequences” that is used for direct pairwise comparison of two nucleotidesequences. “BLAST 2 Sequences” can be accessed and used interactively atthe NCBI website. The “BLAST 2 Sequences” tool can be used for bothblastn and blastp (discussed above).

Percent identity may be measured over the length of an entire definedpolynucleotide sequence or may be measured over a shorter length, forexample, over the length of a fragment taken from a larger, definedsequence, for instance, a fragment of at least 20, at least 30, at least40, at least 50, at least 70, at least 100, or at least 200 contiguousnucleotides. Such lengths are exemplary only, and it is understood thatany fragment length may be used to describe a length over whichpercentage identity may be measured.

A “full length” polynucleotide sequence of a gene is one containing atleast a translation initiation codon (e.g., methionine) followed by anopen reading frame and a translation termination codon. A “full length”polynucleotide sequence encodes a “full length” polypeptide sequence.

A “variant,” “mutant,” or “derivative” of a particular nucleic acidsequence may be defined as a nucleic acid sequence having at least 50%sequence identity to the particular nucleic acid sequence over a certainlength of one of the nucleic acid sequences using blastn with the “BLAST2 Sequences” tool available at the National Center for BiotechnologyInformation's website. (See Tatiana A. Tatusova, Thomas L. Madden(1999), “Blast 2 sequences—a new tool for comparing protein andnucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In someembodiments a variant polynucleotide may show, for example, at least60%, at least 70%, at least 80%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% or greater sequence identity over acertain defined length relative to a reference polynucleotide.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code. It is understood that changes in a nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

A “recombinant nucleic acid” is a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual,2^(nd) ed., vol. 13, Cold Spring Harbor Press, Plainview N.Y. The termrecombinant includes nucleic acids that have been altered solely byaddition, substitution, or deletion of a portion of the nucleic acid.Frequently, a recombinant nucleic acid may include a nucleic acidsequence operably linked to a promoter sequence. Such a recombinantnucleic acid may be part of a vector that is used, for example, totransform a cell.

“Transfection” and “transformation” describe a process by whichexogenous DNA is introduced into a recipient cell. Transfection andtransformation may occur under natural or artificial conditionsaccording to various methods well known in the art, and may rely on anyknown method for the insertion of foreign nucleic acid sequences into aprokaryotic or eukaryotic host cell. The method for transfection ortransformation is selected based on the type of host cell beingtransformed and may include, but is not limited to, bacteriophage orviral infection, electroporation, heat shock, lipofection, and particlebombardment. The terms “transfected cells” and “transformed cells”include stably transfected cells or transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome, as well astransiently transfected cells or transformed cells which express theinserted DNA or RNA for limited periods of time.

The polynucleotide sequences contemplated herein may be present inexpression cassettes and/or expression vectors (e.g., an expressionvector comprising an expression cassette). For example, the vectors maycomprise a polynucleotide encoding an ORF of a recombinant protein(e.g., an exogenous sensor as disclosed herein). The polynucleotidepresent in the vector may be operably linked to a promoter (e.g., aeukaryotic promoter or prokaryotic promoter). “Operably linked” refersto the situation in which a first nucleic acid sequence is placed in afunctional relationship with a second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences may be in close proximity or contiguousand, where necessary to join two protein coding regions, in the samereading frame. Vectors contemplated herein may comprise a heterologouspromoter (e.g., a eukaryotic or prokaryotic promoter) operably linked toa polynucleotide that encodes a protein. A “heterologous promoter”refers to a promoter that is not the native or endogenous promoter forthe protein or RNA that is being expressed. For example, a heterologouspromoter for a LAMP may include a eukaryotic promoter or a prokaryoticpromoter that is not the native, endogenous promoter for the LAMP.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into and mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

As used herein, an “expression cassette” minimally refers to arecombinant polynucleotide comprising a promoter operably linked to arecombinant coding sequence. An expression cassette may be present in avector (e.g., an episomal vector which is transfected into a cell andremains episomal and/or which recombines into the genome of the cell). Avector may include one or more expression cassettes which express one ormore coding sequences (e.g., one or more coding sequences for sensors asdisclosed herein).

The term “vector” refers to some means by which nucleic acid (e.g., DNA)can be introduced into a host organism or host tissue. There are varioustypes of vectors including plasmid vector, bacteriophage vectors, cosmidvectors, bacterial vectors, and viral vectors. As used herein, a“vector” may refer to a recombinant nucleic acid that has beenengineered to express a heterologous polypeptide (e.g., the fusionproteins disclosed herein). The recombinant nucleic acid typicallyincludes cis-acting elements for expression of the heterologouspolypeptide.

Any of the conventional vectors used for expression in eukaryotic cellsmay be used for directly introducing DNA into a subject. Expressionvectors containing regulatory elements from eukaryotic viruses may beused in eukaryotic expression vectors (e.g., vectors containing SV40,CMV, or retroviral promoters or enhancers). Exemplary vectors includethose that express proteins under the direction of such promoters as theSV40 early promoter, SV40 later promoter, metallothionein promoter,human cytomegalovirus promoter, murine mammary tumor virus promoter, andRous sarcoma virus promoter. Expression vectors as contemplated hereinmay include eukaryotic or prokaryotic control sequences that modulateexpression of a heterologous protein (e.g. the fusion protein disclosedherein). Prokaryotic expression control sequences may includeconstitutive or inducible promoters (e.g., T3, T7, Lac, trp, or phoA),ribosome binding sites, or transcription terminators.

The vectors contemplated herein may be introduced and propagated in aprokaryote, which may be used to amplify copies of a vector to beintroduced into a eukaryotic cell or as an intermediate vector in theproduction of a vector to be introduced into a eukaryotic cell (e.g.amplifying a plasmid as part of a viral vector packaging system). Aprokaryote may be used to amplify copies of a vector.

The presently disclosed methods may include delivering one or morepolynucleotides, such as or one or more vectors as described herein, oneor more transcripts thereof, and/or one or proteins transcribedtherefrom, to a host cell. Further contemplated are host cells producedby such methods, and organisms (such as animals, plants, or fungi)comprising or produced from such cells. The disclosed extracellularvesicles may be prepared by introducing vectors that express mRNAencoding a fusion protein as contemplated herein. Conventional viral andnon-viral based gene transfer methods can be used to introduce nucleicacids in mammalian cells or target tissues. Non-viral vector deliverysystems include DNA plasmids, RNA (e.g. a transcript of a vectordescribed herein), naked nucleic acid, and nucleic acid complexed with adelivery vehicle, such as a liposome. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell.

In the methods contemplated herein, a host cell may be transiently ornon-transiently transfected (i.e., stably transfected) with one or morevectors described herein. In some embodiments, a cell is transfected asit naturally occurs in a subject (i.e., in situ). In some embodiments, acell that is transfected is taken from a subject (i.e., explanted). Insome embodiments, the cell is derived from cells taken from a subject,such as a cell line. Suitable cells may include stem cells (e.g.,embryonic stem cells and pluripotent stem cells). A cell transfectedwith one or more vectors described herein may be used to establish a newcell line comprising one or more vector-derived sequences. In themethods contemplated herein, a cell may be transiently transfected withthe components of a system as described herein (such as by transienttransfection of one or more vectors, or transfection with RNA), andmodified through the activity of a complex, in order to establish a newcell line comprising cells containing the modification but lacking anyother exogenous sequence.

A “composition comprising a given polypeptide” and a “compositioncomprising a given polynucleotide” refer broadly to any compositioncontaining the given polynucleotide or amino acid sequence. Thecomposition may comprise a dry formulation or an aqueous solution. Thecompositions may be stored in any suitable form including, but notlimited to, freeze-dried form and may be associated with a stabilizingagent such as a carbohydrate. The compositions may be aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate;SDS), and other components.

“Substantially isolated or purified” nucleic acid or amino acidsequences are contemplated herein. The term “substantially isolated orpurified” refers to nucleic acid or amino acid sequences that areremoved from their natural environment, and are at least 60% free,preferably at least 75% free, and more preferably at least 90% free,even more preferably at least 95% free from other components with whichthey are naturally associated.

The disclosed sensors may include a protease cleavage sequence and/or acognate protease that recognizes and cleaves the protease cleavagesequence. The sensors are not limited to any particular protease orcorresponding protease cleavage site. In some embodiments, the proteaseand cleavage site are from a virus. For example, in certain embodiments,the protease and protease cleavage site are from a virus selected from:tobacco etch virus (TEV), a chymotrypsin-like serine protease andcorresponding cleavage sites, alphavirus proteases and cleavage sites,Hepatitis C virus proteases (e.g., N S3 proteases) and correspondingcleavage sites, chymotrypsin-like cysteine proteases and correspondingcleavage sites, papain-like cysteine proteases and cleavage sites,picornavirus leader proteases and cleavage sites, HIV proteases andcleavage sites, Herpesvirus proteases and cleavage sites, and adenovirusproteases and cleavage sites (see, Tong, Chem. Rev. 2002, 102,4609-4626, herein incorporated by reference in its entirety). Inparticular embodiments, the proteases and cleavage sites are bacterialin original, such as, for example, from Streptomyces griseus protease A(SGPA), SGPB, and alpha-lytic protease and corresponding cleavage sites.In some embodiments, the proteases and cleavage sites are mammalian. Forexample, the proteases could be one of the five major classes ofproteases known in mammals which include serine proteases, cycteineproteases, metallo proteases, aspartic proteases, and thereonineproteases (see, e.g., Turk et al., The EMBO Journal, 2012, 31,1630-1643; Lopez-Otin and Overall, 2002, Nat. Rev. Mol. Cell Biol.,2:509-519; Overall and Blobel, 2007, Nat. Rev. Mol. Cell Biol., 8:245-257; and Lopez-Otin and Bond, 2008, J. Biol. Chem., 283:30422-30437,all of which are herein incorporated in their entireties by references.

The disclosed subject matter relates to engineered red blood cells(eRBCs) which interchangeably may be referred to as red bloodcorpuscles, haematids, erthyroid cells, or erythrocytes. The disclosedeRBCs typically include recombinant membrane proteins for detecting anextracellular ligand and generating a signal if the extracellular ligandis detected (i.e., recombinant exogenous sensors). The disclosedexogenous sensors may be prepared from native and/or non-native proteinsof RBCs.

The disclosed eRBCs are anucleatic (i.e., lack a nucleus) and may beprepared from precursor RBCs (e.g., by inducing precursor RBCs todifferentiate to RBCs). Precursor RBCs may include, but are not limitedto, hemocytoblasts, multipotent heatopoietic stem cells, common myeloidprogenitors, multipotent stem cells, unipotent stem cells,pronormoblasts, proerythroblasts, rubriblasts, normoblasts,erythroblasts, polychromatophilic normoblasts, intermediate normoblasts,orthochromatic normoblasts, and/or late normoblasts. The disclosed eRBCsmay be prepared by transfected precursor RBCs with expression cassettesand/or expression vectors for expressing biosensors as disclosed herein,and subsequently inducing the transfected precursor RBCs todifferentiate to RBCs, thereby obtaining the disclosed eRBCs. Thetransfected precursor RBCs may be induced to differentiate by methodsknown in the art including contacting the transfected precursor RBCsand/or culturing the transfected precursor RBCs with differentiationfactors (e.g., hormones and/or growth factors).

In some embodiments, the recombinant exogenous sensors may be derived,prepared, or engineered from native membrane proteins of RBCs orportions or fragments of membrane proteins of RBCs. Membrane proteins ofRBCs suitable for deriving, preparing, and/or engineering the disclosedsensors may include, but are not limited to glycophorins (e.g.,glycophorin A (GPA or GYPA), glycophorin B (GPB or GYPB), glycophorin C(GPC or GYPC), and/or glycophorin E (GPE or GYPE)); Band 3 (major aniontransporter); Aquaporin 1 (water transporter); Glut1 (glucose andL-dehydroascorbic acid transporter); Kidd antigen protein (ureatransporter); RhAG (gas transporter); Na⁺/K⁺—ATPase; Ca²⁺—ATPase;Na⁺—Cl⁻—cotransporter; Na⁺ K⁺ 2Cl—cotransporter; Na—H exchanger;K—Cl—cotransporter; Gardos channel; ICAM-4; BCAM; Kell antigen (XK);RHD/RhCE; Duffy protein; Adducin; and/or Dematin. In some embodiments,the native membrane protein from which the disclosed recombinantextracellular censors are derived, prepared, and/or engineered is a typeI membrane protein (i.e., single pass membrane protein with anextracellular N-terminus and an intracellular C-terminus such as GPA)and/or a type II membrane protein (i.e., single pass membrane proteinwith an extracellular C-terminus and an intracellular N-terminus such asKell).

The disclosed exogenous sensors include one or more recombinant membraneproteins. In some embodiments, the disclosed exogenous sensors mayinclude two recombinant membrane proteins which are fusion proteinscomprising a membrane protein of a RBC or a portion or fragment thereof.In some embodiments, the fusion proteins may comprise a membrane proteinof a RBC or a portion or fragment thereof to which a ligand bindingdomain has been fused at the N-terminus (i.e., N_(ter)-(ligand bindingdomain)-(RBC membrane protein)-C_(ter)) or at the C-terminus (i.e.,N_(ter)-(RBC membrane protein)-(ligand binding domain)-C_(ter)).Further, the fusion proteins may comprise a membrane protein of a RBC ora portion or fragment thereof to which a functional protein has beenfused at the N-terminus (i.e., N_(ter)-(ligand binding domain)-(RBCmembrane protein)-(functional protein)-C_(ter)) or at the C-terminus(i.e., N_(ter)-(functional protein)-(RBC membrane protein)-(ligandbinding domain)-C_(ter)). A first fusion protein may comprise anon-functional portion of a functional protein, and a second fusionprotein may comprise a second non-functional portion of a functionalprotein such that when the first fusion protein and the second fusionprotein bind the ligand for the ligand binding domain, the firstnon-nonfunctional portion and the second non-functional portion interactto constitute the functional protein.

ILLUSTRATIVE EMBODIMENTS

The following Embodiments are illustrative and should not be interpretedto limit the scope of the claimed subject matter.

Embodiment 1. An engineered red blood cell comprising: (a) a firstexogenous extracellular sensor; the first extracellular sensorcomprising: (i) a first extracellular ligand binding domain (LBD1), (ii)a transmembrane domain (TM1), and (iii) an intracellular first fragmentof a functional protein (FP1), and (b) a second exogenous extracellularsensor; the second extracellular sensor comprising: (i) a secondextracellular ligand binding domain (LBD2), (ii) a transmembrane domain(TM2), and (iii) an intracellular second fragment of the functionalprotein (FP2); wherein: the first extracellular ligand binding domain ofthe first exogenous sensor and the second extracellular ligand bindingdomain of the second exogenous sensor bind to the same ligand to form aternary complex, and the first fragment of the functional protein andthe second fragment of the functional protein interact in the ternarycomplex to reconstitute functional activity of the functional protein(i.e., the ligand comprises binding sites for LBD1 and LBD2).Optionally, TM1 and TM2 may be the same or different and or may comprisea native amino acid sequence of a RBC membrane protein and/or a mutatedamino acid sequence of a RBC membrane protein (e.g., where the nativeamino acid sequence forms multimers and the mutated amino acid sequencedoes not form multimers). Optionally, where FP1 comprises a N-terminalportion of a full-length functional protein and FP2 comprises aC-terminal portion of a full-length functional protein or where FP1comprises a C-terminal portion of a full-length functional protein andFP2 comprises a N-terminal portion of a full-length functional protein.

Embodiment 2. The engineered red blood cell of embodiment 1, wherein thefunctional protein is a fluorescent protein (e.g., a split fluorescentprotein) and the fluorescent protein emits fluorescence when the ternarycomplex is formed and the first fragment of the fluorescent protein andthe second fragment of the fluorescent protein interact to reconstitutethe fluorescent protein.

Embodiment 3. The engineered red blood cell of embodiment 2, wherein thefluorescent protein is selected from the group consisting of greenfluorescent protein (GFP), EGFP (enhanced green fluorescent protein),Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP,ZsGreen, T-Sapphire, GFP-S65T, frGFP, sfGFP, EBFP, EBFP2, Azurite,mTagBFP, ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-IshiCyan, TagCFP, mTFP1 (Teal), Dronpa, EYFP, Topaz, Venus, mCitrine, YPet,TagYFP, PhiYFP, ZsYellow1, mBanana, Kusabira Orange, Kusabira Orange2,mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed,DsRed2, DsRed-Express (T1), DsRed-Monomer, mTangerine, mRuby, mApple,mStrawberry, AsRed2, mRFP1, JRed, mCherry, mKate, HcRed1, mRaspberry,dKeima-Tandem, HcRed-Tandem, mPlum, and AQ143.

Embodiment 4. The engineered red blood cell of any of the foregoingembodiments, wherein the first extracellular ligand binding domain(LBD1) and the transmembrane domain of the first exogenous extracellularsensor (TM1) are linked by a 5-25 amino acid linking sequence of aminoacids selected from glycine, serine, and combination thereof (e.g.,LBD1-GGGSGGGS-TM1); and/or wherein the transmembrane domain (TM1) andthe intracellular first fragment of the functional protein of the firstexogenous extracellular sensor (FP1) are linked by a 5-25 amino acidlinking sequence of amino acids selected from glycine, serine, andcombination thereof (e.g., TM1-GGGSGGGS-FP1).

Embodiment 5. The engineered red blood cell of any of the foregoingembodiments, wherein the second extracellular ligand binding domain(LBD2) and the transmembrane domain of the second exogenousextracellular sensor (TM2) are linked by a 5-25 amino acid linkingsequence of amino acids selected from glycine, serine, and combinationthereof (e.g., LBD2-GGGSGGGS-TM2); and/or wherein the transmembranedomain TM2 and the intracellular first fragment of the functionalprotein of the second exogenous extracellular sensor (FP2) are linked bya 5-25 amino acid linking sequence of amino acids selected from glycine,serine, and combination thereof (e.g., TM2-GGGSGGGS-FP2).

Embodiment 6. The engineered red blood cell of any of the foregoingembodiments, wherein the functional protein is an enzyme and the enzymeexhibits enzymatic activity emits when the ternary complex is formed andthe first fragment of the fluorescent protein and the second fragment ofthe enzyme interact to reconstitute the enzyme.

Embodiment 7. The engineered red blood cell of embodiment 6, wherein theenzyme is a luciferase.

Embodiment 8. A combination of expression cassettes for preparing anengineered red blood cell, the combination comprising: (a) a firstcassette expressing a first exogenous extracellular sensor; the firstextracellular sensor comprising: (i) a first extracellular ligandbinding domain (LBD1), (ii) a transmembrane domain (TM1), and (iii) anintracellular first fragment of a functional protein (FP1), and (b) asecond cassette expressing a second exogenous extracellular sensor; thesecond extracellular sensor comprising: (i) a second extracellularligand binding domain (LBD2), (ii) a transmembrane domain (TM2), and(iii) an intracellular second fragment of the functional protein (FP2);wherein: the first extracellular ligand binding domain of the firstexogenous sensor and the second extracellular ligand binding domain ofthe second exogenous sensor bind to the same ligand to form a ternarycomplex, and the first fragment of the functional protein and the secondfragment of the functional protein interact in the ternary complex toreconstitute functional activity of the functional protein (i.e., theligand comprises binding sites for LBD1 and LBD2). Optionally, TM1 andTM2 may be the same or different and or may comprise a native amino acidsequence of a RBC membrane protein and/or a mutated amino acid sequenceof a RBC membrane protein (e.g., where the native amino acid sequenceforms multimers and the mutated amino acid sequence does not formmultimers). Optionally, FP1 comprises an N-terminal portion of afull-length functional protein and FP2 comprises a C-terminal portion ofa full-length functional protein or where FP1 comprises a C-terminalportion of a full-length functional protein and FP2 comprises anN-terminal portion of a full-length functional protein.

Embodiment 9. The combination of embodiment 8, wherein the expressioncassettes are present on separate vectors.

Embodiment 10. The combination of embodiment 8, wherein the expressioncassettes are present on the same vector.

Embodiment 11. The combination of any of the foregoing embodiments,wherein the functional protein is a fluorescent protein (e.g., a splitfluorescent protein) and the fluorescent protein emits fluorescence whenthe ternary complex is formed and the first fragment of the fluorescentprotein and the second fragment of the fluorescent protein interact toreconstitute the fluorescent protein.

Embodiment 12. The combination of embodiment 11, wherein the fluorescentprotein is selected from the group consisting of green fluorescentprotein (GFP), EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi,TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, GFP-S65T, frGFP, sfGFP,EBFP, EBFP2, Azurite, mTagBFP, ECFP, mECFP, Cerulean, mTurquoise, CyPet,AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal), Dronpa, EYFP, Topaz,Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, KusabiraOrange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem,TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer,mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry,mKate, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, andAQ143.

Embodiment 13. The combination of any of the foregoing embodiments,wherein the first extracellular ligand binding domain (LBD1) and thetransmembrane domain of the first exogenous extracellular sensor (TM1)are linked by a 5-25 amino acid linking sequence of amino acids selectedfrom glycine, serine, and combination thereof (e.g., LBD1-GGGSGGGS-TM1);and/or wherein the transmembrane domain (TM1) and the intracellularfirst fragment of the functional protein of the first exogenousextracellular sensor (FP1) are linked by a 5-25 amino acid linkingsequence of amino acids selected from glycine, serine, and combinationthereof (e.g., TM1-GGGSGGGS-FP1).

Embodiment 14. The combination of any of the foregoing embodiments,wherein the second extracellular ligand binding domain (LBD2) and thetransmembrane domain of the second exogenous extracellular sensor (TM2)are linked by a 5-25 amino acid linking sequence of amino acids selectedfrom glycine, serine, and combination thereof (e.g., LBD2-GGGSGGGS-TM2);and/or wherein the transmembrane domain TM2 and the intracellular firstfragment of the functional protein of the second exogenous extracellularsensor (FP2) are linked by a 5-25 amino acid linking sequence of aminoacids selected from glycine, serine, and combination thereof (e.g.,TM2-GGGSGGGS-FP2).

Embodiment 15. The combination of any of the foregoing embodiments,wherein the functional protein is an enzyme and the enzyme exhibitsenzymatic activity emits when the ternary complex is formed and thefirst fragment of the fluorescent protein and the second fragment of theenzyme interact to reconstitute the enzyme.

Embodiment 16. The combination of embodiment 15, wherein the enzyme is aluciferase.

Embodiment 17. A method for detecting a ligand (or a metabolite), themethod comprising contacting the engineered red blood cell of any ofembodiments 1-7 with the ligand (or metabolite), and detectingfunctional activity of the functional protein.

Embodiment 18. The method of embodiment 17, wherein the functionalprotein is a fluorescent protein and detecting functional activitycomprises detecting fluorescence.

Embodiment 19. The method of embodiment 17, wherein the functionalprotein is a luciferase protein and detecting functional activitycomprises contacting the engineered red blood cell with a substrate forthe luciferase protein and detecting light emitted from the engineeredred blood cell.

Embodiment 20. A method for preparing the engineered red blood cells ofany of embodiments 1-7, the method comprising: (I) transfecting aprecursor red blood cell with a combination of expression cassettes ofany of embodiments 8-16; and (II) inducing the transfected precursor redblood cell precursor cell to differentiate into a red blood cell,thereby preparing the engineered red blood cell.

Embodiment 21. A precursor red blood cell transfected with the cassettesof any of embodiments 8-16.

Embodiment 22. The engineered red blood cells of any of embodiments 1-7,wherein the first exogenous extracellular sensor and/or the secondexogenous extracellular sensor comprise an amino acid sequence of anative membrane protein of red blood cells or a portion or fragmentthereof (e.g., at least a portion of the transmembrane amino acidsequence of a native membrane protein of red blood cells, optionally atleast a portion of the extracellular amino acid sequence of a nativemembrane protein of red blood cells, and optionally at least a portionof the intracellular amino acid sequence of a native membrane protein ofred blood cells).

Embodiment 23. The combination of expression cassettes of any of claims8-16, wherein the first exogenous extracellular sensor and/or the secondexogenous extracellular sensor comprise an amino acid sequence of anative membrane protein of red blood cells or a portion or fragmentthereof (e.g., at least a portion of the transmembrane amino acidsequence of a native membrane protein of red blood cells, optionally atleast a portion of the extracellular amino acid sequence of a nativemembrane protein of red blood cells, and optionally at least a portionof the intracellular amino acid sequence of a native membrane protein ofred blood cells).

Embodiment 24. The method of any of embodiments 17-20, wherein the firstexogenous extracellular sensor and/or the second exogenous extracellularsensor comprise an amino acid sequence of a native membrane protein ofred blood cells or a portion or fragment thereof (e.g., at least aportion of the transmembrane amino acid sequence of a native membraneprotein of red blood cells, optionally at least a portion of theextracellular amino acid sequence of a native membrane protein of redblood cells, and optionally at least a portion of the intracellularamino acid sequence of a native membrane protein of red blood cells).

Embodiment 25. The precursor red blood cell of embodiment 21, whereinthe first exogenous extracellular sensor and/or the second exogenousextracellular sensor comprise an amino acid sequence of a nativemembrane protein of red blood cells or a portion or fragment thereof(e.g., at least a portion of the transmembrane amino acid sequence of anative membrane protein of red blood cells, optionally at least aportion of the extracellular amino acid sequence of a native membraneprotein of red blood cells, and optionally at least a portion of theintracellular amino acid sequence of a native membrane protein of redblood cells).

EXAMPLES

The following Examples are illustrative and should not be interpreted tolimit the scope of the claimed subject matter.

Example 1

Abstract

Cell-based therapies comprise a blossoming, multi-billion dollarindustry addressing markets ranging from cancer to autoimmune disease.An emerging frontier is the use of red blood cells (RBC), which haveexceptionally long circulation times (up to 120 days—far longer thansynthetic vehicles), lack DNA (and thus are safe), and can be loadedwith drugs, proteins, or other cargo. Technologies that enable one toengineer RBCs to perform specific functions in vivo could serve unmetdiagnostic and therapeutic needs. This invention comprises a technologyfor generating engineered RBC (eRBC) to serve as long-lived biosensors.Each eRBC would emit a fluorescent readout when bound to the analyte ofinterest, such that eRBC state could be monitored non-invasively usingestablished technologies for fluorescent imaging (e.g., fluorescentimaging of the retina).

Applications

Applications for the disclosed technology may include, but are notlimited to: (a) Diagnostics: using simple imaging technology, a patientcould perform regular self-analysis and enable real-time, high frequencymonitoring outside clinical settings, none of which is possible withexisting technologies requiring specialized equipment, trainedpersonnel, and/or sample collection; (b) Exposure monitoring: by asimilar approach, military personal and first responders could determinewhether or not they have been exposed to a potential infectious agentvia monitoring output from a tailored eRBC; and (c) Basic science: eRBCcould provide a facile approach to non-invasively monitoring solublespecies in the serum of experimental animals.

Advantages

Advantages of the disclosed technology may include, but are not limitedto: (a) eRBC are longer-lived than any injectable particle or smallmolecule; a single injection could enable monitoring for weeks ormonths; (b) eRBC biosensor output is amenable to noninvasive monitoring,without requiring sample collection, complicated analysis, or trainedpersonnel; (c) the disclosed protein biosensors are readily customizedto detect novel analytes—all that is needed is replacing the ligandbinding domain with binding proteins (e.g., single chain antibodies)that bind the analyte of interest; and (c) RBC have been engineered forother applications but this represents the first technology enabling theengineering of eRBC to serve as biosensors for a range of analytes foundin the blood.

Brief Summary of the Technology

This technology comprises a protein biosensor platform for engineeringRBCs such that sensing of an extracellular analyte results in thereconstitution of a split functional protein in the RBC cytosol. Thebiosensor comprises two protein chains, each of which includes anextracellular ligand binding domain, a membrane-spanning domain, and anintracellular domain comprising half of a split functional protein (FIG.1). In the proposed mechanism, upon binding of the ligand to the ligandbinding domains, the two chains dimerize, bringing the two split proteinhalves into proximity causing the split functional protein toreconstitute and therefore exhibit functionality. Suitable functionalproteins may include, but are not limited to, fluorescent proteins andenzymatic proteins.

In FIG. 1, each biosensor is composed of two protein chains each with anextracellular ligand binding domain (LBD1 and LBD2, respectively) and asingle transmembrane pass domain (TM1 and TM2, respectively). Theintracellular portion of the receptors contains one half of a splitfluorescent protein (FP1 and FP2, respectively). Upon ligand binding,the two chains dimerize, allowing to two split protein halves to comeinto contact and reconstitute the fluorescent protein and then emitfluorescence.

Technical Description

As an initial proof of concept, we developed a receptor withextracellular ligand binding domains that heterodimerize upon bindingthe small molecule rapamycin (FRB and FKBP form a heterodimer in thepresence of rapamycin) (FIG. 1) (for a review of the FKBP-Rapamycin-FRBternary complex, see Banaszynski, et al., J. Am. Chem. Soc. (JACS)Articles, Mar. 9, 2005, 127, 4715-4721, the content of which isincorporate herein by reference in its entirety). In this preliminarywork, we used split GFP (sGFP) as the split fluorescent protein [Ghosh2000], although the eventual application of this technology in vivowould probably utilize reconstitution of a split infrared fluorescentprotein [Filonov 2013] or split luciferase [Dixon 2016]. Our initialconstructs utilized a simple single-pass transmembrane domain, and noneof these receptors exhibited ligand-inducible reconstitution of GFP(FIG. 2).

The results in FIG. 2 represent experiments in which a library ofreceptors encoding a rapamycin binding domain, transmembrane domain, andsplit GFP half was created and transfected in pair-wise combinationsinto HEK293FT cells. The background signaling was very low, with mostcells actually showing GFP values at or below that of the control cells(transfected with only one receptor chain). Furthermore, no combinationof receptors, regardless of linker length, showed any induction of GFPupon addition of rapamycin. All transfections were performed using theCaCl₂-HEPES buffered saline methodology. Sixteen (16) hours posttransfection, rapamycin was added with media change, and the cells wereallowed to incubate for an additional 24 hours. Cells were harvested andanalyzed by flow cytometry.

We next investigated whether biosensors could be constructed by fusingligand-binding and sGFP domains to the RBC-resident proteins Kell andGlycophorin A (GPA) (FIG. 3). Kell and GPA are expressed in RBC evenwhen expressed as genetic fusion constructs [Shi 2014]. Moreover, sinceGPA is a Type I membrane protein and Kell is a Type II membrane protein(Kell and GPA are expressed in opposite orientation relative to theplasma membrane), we hypothesized that fusion of sGFP fragments to theintracellular terminus of each protein may bring the sGFP fragmentstogether in the antiparallel orientation required for reconstitution ofGFP. As illustrated in FIG. 3, Kell and GPA were modified to containextracellular rapamycin-binding domains (RBD1 and RBD2 at theirextracellular N-terminus and extracellular C-terminus, respectively) andintracellular split GFP halves (GFP1 and GFP2 at their intracellularC-terminus and intracellular C-terminus, respectively). Upon theaddition of rapamycin, these two protein chains can dimerize, allowingfor the reconstitution of GFP. These two proteins are naturallyantiparallel to one another: Kell possesses an extracellular C-terminus,and GPA an extracellular N-terminus.

Several of these second-generation biosensors exhibited ligand-induciblereconstitution of fluorescence (FIG. 4). Because only some combinationsof receptor designs conferred ligand-inducible reconstitution, wehypothesized that geometric constraints may limit reconstitution inthese receptors. Thus, we introduced longer linkers into theintracellular portions of these functional receptors (i.e.,glycine-serine linkers between the transmembrane domain portion andfunctional protein portion of the sensors), and we observed thatligand-induced reconstitution was indeed dramatically improved (FIG. 4).

The results in FIG. 4 represent experiments in which pairs of receptorswere expressed in HEK 293FT cells. These data indicate that splitprotein reconstitution on a membrane is feasible, given that thereceptors achieve the necessary orientation for protein reconstitution.All transfections were performed using the CaCl₂-HEPES buffered salinemethodology. Sixteen (16) hours post-transfection, rapamycin was addedalong with media change, and cells were incubated for an additional 24hours before being harvested and analyzed by flow cytometry.

These initial experiments demonstrated that RBC-resident proteins may beengineered into ligand-inducible biosensor proteins, and that suchproteins may be iteratively improved using design-driven approaches.These data also support the feasibility of adapting this modularreceptor design to incorporate novel ligand binding domains and novelfluorophore outputs

Because GPA is known to be expressed at higher levels than is Kell, wealso investigated GPA-only biosensors (FIG. 5). As illustrated in FIG.5, GPA, a native RBC protein, was modified to create two sensors (i.e.,two fusion proteins) having extracellular rapamycin-binding domains (FRBor FKBP) and intracellular split GFP halves (GFPS and GFPL). Upon theaddition of rapamycin, these two protein chains can dimerize, allowingfor the reconstitution of GFP. These biosensors also conferredligand-inducible reconstitution of sGFP (FIG. 6).

The results in FIG. 6 represent experiments in which pairs of receptorswere expressed in HEK 293FT cells. All transfections were performedusing the CaCl2-HEPES buffered saline methodology. Sixteen (16) hourspost-transfection, rapamycin was added along with media change, andcells were incubated for an additional 24 hours before being harvestedand analyzed by flow cytometry.

We hypothesized that biosensors based upon wild-type GPA exhibited highbackground signaling due to homodimerization in the absence of ligand,and therefore we investigated biosensors in which one or both GPAdomains required for homodimerization were mutated to reducehomodimerization in the absence of ligand (FIG. 7). As illustrated inFIGS. 7-9, biosensor proteins described in FIG. 6 were modified tomutate the transmembrane domain of GPA to decrease native backgrounddimerization. As illustrated in FIG. 7 and FIG. 8, GPA was mutated inthe transmembrane domain (GPAm) by converting the neutral valine atposition 84 to a charged amino acid (V84R: Valine 84 to Arginine, V84E:Valine 84 to Glutamic Acid, V84K: Valine 84 to Lysine), and GPA wasmodified to contain N-terminal extracellular rapamycin-binding domains(FRB or FKBP) and C-terminal intracellular split GFP halves (GFPS orGFPL). Upon the addition of rapamycin, these two protein chains candimerize, allowing for the reconstitution of GFP.

The mutated proteins then were expressed in HEK293FT cells to testdimerization in the presence of rapamycin. (See FIG. 8 and FIG. 9). Theresults in FIG. 8 and FIG. 9 represent experiments in which pairs ofreceptors were expressed in HEK 293FT cells. All transfections wereperformed using the CaCl₂-HEPES buffered saline methodology. Sixteen(16) hours post-transfection, rapamycin was added along with mediachange, and cells were incubated for an additional 24 hours before beingharvested and analyzed by flow cytometry. As observed, the mutatedbiosensors exhibited sGFP reconstitution that was lower that thatexhibited by wild-type GPA based receptors but higher than thatexhibited by Kell-based biosensors (FIG. 8 and FIG. 9).

Because sGFP reconstitution efficiency may be limited by parameters notdirectly related to the eRBC biosensor mechanism, we also investigatedwhether we could generate a biosensor based upon reconstitution of adifferent signaling domain—split NANOLUCIFERASE™ [Dixon 2016] (FIG. 10).As illustrated in FIG. 10, GPA was mutated in the transmembrane domain(GPAm, see FIGS. 7-9) and was modified to contain N-terminalextracellular rapamycin-binding domains (FRB or FKBP) and C-terminalintracellular split NANOLUCIFERASE™ halves (114 and 11S). Upon theaddition of rapamycin, these two protein chains can dimerize, allowingfor the reconstitution of NANOLUCIFERASE™, and luminescence uponaddition of the substrate, furimazine.

These NANOLUCIFERASE™-based biosensors exhibited robust ligand-induciblereconstitution of luciferase activity, when expressed in HEK293FT cells(FIG. 11). The results in FIG. 11 represent experiments in which pairsof receptors were expressed in HEK 293FT cells. All transfections wereperformed using the CaCl₂-HEPES buffered saline methodology. Sixteen(16) hours post-transfection, rapamycin was added along with mediachange, and cells were incubated for an additional 24 hours before beingharvested and analyzed by plate reader for luciferase luminescence.

To test whether such eRBC biosensors would be expressed in functional inred blood cells (erythrocytes), we next expressed our biosensors in G1ercells. G1er cells can be induced to differentiate into erythrocytes byexposure to beta-estradiol. We observed that our biosensors were highlyfunctional in G1er cells, without or without differentiation stimuli,and when differentiated for various lengths of time (FIG. 12). Asillustrated in FIG. 12, G1er cells were spinoculated with retroviruscontaining pairs of receptors and GFP expressed in one plasmid. Cellswere sorted for GFP expression. GFP+ cells received either ethanol or-estradiol (to drive differentiation into an erythrocyte phenotype) andeither DMSO or rapamycin. Cell lysates were collected in passive lysisbuffer 24 hours and 48 hours after receiving treatment. Technicalreplicates of cell lysates were analyzed for GFP fluorescence on a platereader. Then a luciferase assay was performed on the cell lysates withthe addition of substrate. Abbreviations: FD—fold difference. Theseresults indicate that our eRBC biosensors are expressed, retained, andfunctional in erythrocytes, validating the core concept described inthis disclosure.

We also tested the biosensors in human CD34+ cells. The results in FIG.13 represent experiments in which human CD34+ cells were spinoculatedwith a lentiviral vector expressing a pair of receptors and Puromycinresistance. Cells were selected with Puromycin and then differentiatedfor 14 days. Differentiated cells received either DMSO or rapamycin. Onehour after treatment, cell lysates were collected in passive lysisbuffer. A luciferase assay was performed on cell lysates.

REFERENCES

Shi, J. H. et al. Engineered red blood cells as carriers for systemicdelivery of a wide array of functional probes. Proceedings of theNational Academy of Sciences of the United States of America 111,10131-10136, doi:DOI 10.1073/pnas.1409861111 (2014).

Ghosh, I., Hamilton, A. D. & Regan, L. Antiparallel leucinezipper-directed protein reassembly: Application to the green fluorescentprotein. J Am Chem Soc 122, 5658-5659, doi:Doi 10.1021/Ja994421w (2000).

Filonov, G. S., Verkhusha, V. V. A Near-Infrared BiFC Reporter for InVivo Imaging of Protein-Protein Interactions. Chemistry & Biology 20,1078-1086 (2013).

Daringer, N; Dudek, R; Schwarz, K; Leonard, J. A Modular ExtracellularSensor Architecture for Engineering Mammalian Cell-based Devices. ACSSynthetic Biology, 3 (12), 892-902 (2014).

Dixon, A. S., et al. Nantou Complementation Reporter Optimized forAccurate Measurement of Protein Interactions in Cells. ACS Chem Biol11(2) 400-408, doi: DOI 10.1021/acschembio.5b00753 (2016).

Li, E., et al. Transmembrane helix dimerization: beyond the search forsequence motifs. Biochim Biophys Acta 1818(2): 183-193 (2012).

Lemmon, M. A., et al. Glycophorin A dimerization is driven by specificinteractions between transmembrane alpha-helices. J Biol Chem 267(11):7683-7689 (1992).

Example 2

Reference is made to the Abstract entitled “Engineering Red BloodCell-Based Biosensors for Physiological Monitoring,” Authors: Dolberg,T. B., Schwarz, K. A., Leonard, J. M. which was presented at the 2016Synthetic Biology: Engineering, Evolution & Design (SEED) conference onJul. 19, 2016, and which Abstract is incorporated by reference herein inits entirety.

Cell-based therapies have a wide range of applications ranging fromcancer immunotherapy to regenerative medicine. A promising emergingfrontier of this field is the development of engineered red blood cells(eRBCs) for therapeutic and diagnostic applications. RBCs haveexceptionally long circulation times (around 40 days—far longer thansynthetic vehicles), lack DNA (and thus are safe), and can be loadedwith drugs, proteins, or other cargo. Technologies that enable one toengineer RBCs to perform specific functions in vivo could serve unmetdiagnostic and therapeutic needs. In particular, new technologies arerequired for non-invasive, routine monitoring for pathogen exposure(e.g., in the context of first responders) and for “actionable” analytes(e.g., markers of inflammation post-surgery).

In this project, we sought to develop eRBC biosensors than detect highlytoxic agents, with the long-term goal of enabling one to detect exposureto these agents prior to the onset of physical symptoms. As a first steptowards this goal, we designed and evaluated a novel biosensor strategythat is suitable to achieving biosensing in eRBCs, which lack DNA andthus require a readout other than gene expression. Towards this end, weengineered a novel cell-surface receptor protein in which ligand bindinginduces receptor dimerization, which then facilitates reconstitution ofan intracellular split fluorescent protein. Ultimately, eRBCfluorescence may be monitored noninvasively using establishedtechnologies for fluorescent imaging of the retina. Importantly, ourstrategy involves modification of RBC-resident proteins, since retentionof membrane proteins during RBC maturation is a tightly regulated and anincompletely understood process. In this study, we comparativelyevaluated a range of biosensor architectures that implement the proposedmechanism, identified design biosensor features that successfullyconferred significant ligand-induced generation of fluorescent output,and investigated strategies for improving biosensor performance (e.g.,minimization of background fluorescence and enhancing fold-inductionupon exposure to ligand). This crucial proof-of-principle demonstrationestablishes a foundation for developing eRBC biosensors that couldultimately address an unmet need for noninvasive monitoring ofphysiological signals for a range of diagnostic applications.

Example 3

Reference is made to the Abstract entitled “Engineering Red BloodCell-Based Biosensors for Physiological Monitoring,” Authors: Dolberg,T. B., Schwarz, K. A., Leonard, J. M., which was presented at the 2016annual conference of the American Institute of Chemical Engineers(AlChE) on Nov. 14, 2016 and which Abstract is incorporated by referenceherein in its entirety.

Cell-based therapies have a wide range of applications ranging fromcancer immunotherapy to regenerative medicine. A promising emergingfrontier of this field is the development of engineered red blood cells(eRBCs) for therapeutic and diagnostic applications. RBCs haveexceptionally long circulation times (around 120 days—far longer thansynthetic vehicles), lack DNA (and thus are safe), and can be loadedwith drugs, proteins, or other cargo. Technologies that enable one toengineer RBCs to act as biosensors, performing specific functions invivo, could serve unmet diagnostic and therapeutic needs. In particular,new technologies are required for non-invasive, routine monitoring forpathogen exposure (e.g., in the context of first responders) and forother “actionable” analytes (e.g., markers of inflammationpost-surgery).

In this project, we are developing eRBC biosensors that generate afluorescent output upon detection of the analyte of interest.Ultimately, eRBC biosensor fluorescent output may be monitorednon-invasively using established technologies for fluorescent imaging ofthe retina. Using this simple imaging technology, a patient couldperform regular self-analysis and enable real time, high frequencymonitoring outside clinical settings, none of which is possible withexisting technologies requiring specialized equipment, trainedpersonnel, and/or sample collection. Thus, such biosensors that enablethe detection of actionable analytes could benefit exposed personnel byaccelerating the initiation of treatment (perhaps before obvioussymptoms present) and reduction of further exposure risks when possible.

As a first step towards the goal of building eRBC biosensors, wedesigned and evaluated a novel biosensor strategy that is suitable forachieving biosensing in eRBCs, which lack DNA and thus require a readoutother than gene expression. Towards this end, we engineered a novelcell-surface receptor protein in which ligand binding induces receptordimerization, which then facilitates reconstitution of an intracellularsplit fluorescent protein. Importantly, our strategy involvesmodification of RBC-resident proteins, since retention of membraneproteins during RBC maturation is a tightly regulated and anincompletely understood process. We comparatively evaluated a range ofbiosensor architectures that implement the proposed mechanism, enablingus to identify biosensor designs and design features that successfullyconferred significant ligand-induced generation of fluorescent output.We also evaluated and implemented strategies for improving biosensorperformance, including minimization of background fluorescence andenhancing fold-induction upon exposure to ligand. This crucialproof-of-principle demonstration establishes a foundation for developingeRBC biosensors that could ultimately address an unmet need fornon-invasive monitoring of physiological signals for a range ofdiagnostic applications.

Example 4

Reference is made to the Abstract entitled “Engineering Red BloodCell-Based Biosensors for Physiological Monitoring,” Authors: Dolberg,T. B., Schwarz, K. A., Leonard, J. M.,” which is to be presented at the2017 Midwest Regional Conference (MRC) of the American Institute ofChemical Engineers (AlChE) on Mar. 1, 2017 and which Abstract isincorporated by reference herein in its entirety.

Cell-based therapies have a wide range of applications ranging fromcancer immunotherapy to regenerative medicine. A promising emergingfrontier of this field is the development of engineered red blood cells(eRBCs) for therapeutic and diagnostic applications. RBCs are anattractive platform for diagnostics because they have exceptionally longcirculation times (around 120 days—far longer than synthetic vehicles),lack DNA (and thus are safe), and can be loaded with drugs, proteins, orother cargo. Recent technological advances have enabled the large-scaleproduction of RBCs from precursor cells, which may potentially beharnessed to generate off-the shelf eRBC-based products to meet medicalneeds, including both diagnostic and therapeutic applications.

The specific goal of this project is to generate eRBC-based technologiesenabling noninvasive monitoring for pathogen exposure (e.g., in thecontext of first responders) and for other “actionable” analytes (e.g.,markers of inflammation post-surgery). Towards this goal, we aredeveloping eRBC biosensors that generate a fluorescent output upondetection of the analyte of interest, and this output may be monitorednon-invasively using established technologies for fluorescent imaging ofthe retina. These biosensors would enable the detection of actionableanalytes thus benefitting exposed personnel by accelerating theinitiation of treatment (perhaps before obvious symptoms present) andreducing of further exposure risks when possible.

As a first step to enable RBCs to act as sensors, we designed andevaluated a novel biosensor strategy that is suitable for achievingbiosensing in eRBCs, which lack DNA and thus require a readout otherthan gene expression. Towards this end, we engineered a novelcell-surface receptor protein in which ligand binding induces receptordimerization, which then facilitates reconstitution of an intracellularsplit fluorescent protein. Importantly, our strategy involvesmodification of RBC-resident proteins, since retention of membraneproteins during RBC maturation is a tightly regulated and anincompletely understood process. We comparatively evaluated a range ofbiosensor architectures that implement the proposed mechanism, enablingus to identify biosensor designs and design features that successfullyconferred significant ligand-induced generation of fluorescent output.We also evaluated and implemented strategies for improving biosensorperformance, including minimization of background fluorescence andenhancing fold-induction upon exposure to ligand. This crucialproof-of-principle demonstration establishes a foundation for developingeRBC biosensors that could ultimately address an unmet need fornon-invasive monitoring of physiological signals for a range ofdiagnostic applications.

Example 5

Reference is made to U.S. Publication No. 2014-0234851, published onAug. 21, 2014, which discloses modular extracellular sensor architecture(MESA) for cell-based biosensors, and which is incorporated herein byreference in its entirety. The modular exogenous extracellular sensorarchitecture disclosed in U.S. Publication No. 2014-0234851 may beadapted for use in the engineered red blood cell-based biosensorsdisclosed and contemplated herein.

Example 6

Background. This example describes how engineered mammalian biosensortechnology can be applied to RBC-based technologies. The MESA family ofengineered cell-surface biosensors (see, e.g., U.S. Publication No.2014-0234851) enables the transduction of an extracellularligand-binding event into a change in intracellular state. Of particularrelevance to engineering RBCs, this intracellular state change cancomprise reconstitution of a split protein, which may be an enzyme (asdemonstrated using a split TEV protease) or potentially anotherfunctional domain (e.g., a split fluorescent protein). Brieflysummarized below are several potential applications for engineering highvalue RBC using such technologies.

Pathogen exposure monitoring. RBCs could be engineered to monitorexposure to a pathogen and provide a readout that is easily monitored. Atarget pathogen could be dengue virus, for which serial exposure tomultiple serotypes significantly increases risk for developing severehemorrhagic symptoms. Thus, using engineered RBCs (eRBCs) to monitorpatients or warfighters for secondary exposure could trigger rapidintervention to reduce morbidity or mortality. The biosensor readoutcould be reconstitution of a split fluorescent protein (e.g., aninfrared fluorescent protein (IFP) that is spectrally distinguishablefrom hemoglobin in RBC), and eRBC fluorescence could be monitored usinga technique similar to fluorescent angiography of the retina. Such eRBCscould also incorporate a second fluorophore to enable quantitativenormalization of biosensor readout. Distinct biosensors could beengineered to provide pathogen or serotype-specific biosensing.Alternatively, biosensor readout could comprise reconstitution of abioluminescent catalyst (e.g., luciferase or another enzyme, for examplethat catalyzes production of a product that yields a color change inexcreted urine) and/or eRBC biosensor state could be evaluated ex vivousing a blood sample.

Physiological monitoring. Using techniques similar to those describedabove, eRBC biosensors could be used to monitor blood concentrations ofphysiologically-important species over time using noninvasivetechniques. Potential targets include (a) Low-density lipoprotein (LDL),certain forms of which promote immuno-inflammatory processes and driveatherosclerosis, which could be useful for evaluating or trackingcholesterol levels or (b) cytokines that predict the onset of acutetransplant rejection, such as IL-6, which may predict acute kidneytransplant rejection.

Incorporation of MESA-type biosensors into eRBCs. Because only a subsetof proteins is retained in mature RBC, MESA-type biosensors may beredesigned to coopt localization of retained proteins such asglycophorin or Kell. Potential strategies include non-covalent tetheringvia the introduction of protein-protein interaction domains into bothMESA proteins and glycophorin and/or Kell.

Induction of immune tolerance. Although expression of non-native proteindomains on the surface of eRBC may enable immunological clearance ofeRBCs, some evidence indicates that expression of proteins in or on RBCpromotes active induction of immunological tolerance.

In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references may be madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A biosensor comprising: (a) a first protein comprising: (i)a first extracellular ligand binding domain, (ii) a first transmembranedomain, (iii) an intracellular first fragment of a functional protein,and (iv) an amino acid linker connecting the first transmembrane domainand the intracellular first fragment of the functional protein; and (b)a second protein comprising: (i) a second extracellular ligand bindingdomain, (ii) a second transmembrane domain, (iii) an intracellularsecond fragment of the functional protein, and (iv) an amino acid linkerconnecting the second transmembrane domain and the intracellular secondfragment of the functional protein; wherein the first extracellularligand binding domain and the second extracellular ligand binding domainbind to the same ligand, wherein the first fragment of the functionalprotein and the second fragment of the functional protein are capable ofinteracting to reconstitute activity of the functional protein, andwherein the amino acid linker of the first protein or the amino acidlinker of the second protein comprises 7-25 amino acids.
 2. Thebiosensor of claim 1, wherein the amino acid linker of the first proteinand the amino acid linker of the second protein each comprise 7-25 aminoacids.
 3. The biosensor of claim 1, wherein the amino acid linker of thefirst protein and the amino acid linker of the second protein eachcomprise a sequence of amino acids selected from glycine, serine, andcombinations thereof.
 4. The biosensor of claim 1, wherein the aminoacid linker of the first protein and the amino acid linker of the secondprotein each comprise the sequence GGGSGGGS.
 5. The biosensor of claim1, wherein the functional protein is a fluorescent protein and thefluorescent protein emits fluorescence when the ternary complex isformed and the first fragment of the fluorescent protein and the secondfragment of the fluorescent protein interact to reconstitute thefluorescent protein.
 6. The biosensor of claim 5, wherein thefluorescent protein is selected from the group consisting of greenfluorescent protein (GFP), EGFP, Emerald, Superfolder GFP, Azami Green,mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, GFP-S65T, frGFP,sfGFP, EBFP, EBFP2, Azurite, mTagBFP, ECFP, mECFP, Cerulean, mTurquoise,CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal), Dronpa, EYFP,Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana,Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato,dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1),DsRed-Monomer, mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFP1,JRed, mCherry, mKate, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem,mPlum, and AQ143.
 7. The biosensor of claim 1, wherein the firstextracellular ligand binding domain and the first transmembrane domainof the first recombinant extracellular sensor are linked by a 5-25 aminoacid sequence of amino acids selected from glycine, serine, andcombinations thereof.
 8. The biosensor of claim 1, wherein the secondextracellular ligand binding domain and the second transmembrane domainof the first recombinant extracellular sensor are linked by a 5-25 aminoacid sequence of amino acids selected from glycine, serine, andcombinations thereof.
 9. The biosensor of claim 1, wherein the firstextracellular ligand binding domain and the first transmembrane domainof the first recombinant extracellular sensor are linked by a 5-25 aminoacid sequence of amino acids selected from glycine, serine, andcombinations thereof; and wherein the second extracellular ligandbinding domain and the second transmembrane domain of the firstrecombinant extracellular sensor are linked by a 5-25 amino acidsequence of amino acids selected from glycine, serine, and combinationsthereof.
 10. The biosensor of claim 1, wherein the functional protein isan enzyme and the enzyme exhibits enzymatic activity when the ternarycomplex is formed and the first fragment of the enzyme and the secondfragment of the enzyme interact to reconstitute the enzyme.
 11. Thebiosensor of claim 10, wherein the enzyme is a luciferase.
 12. Acombination of expression cassettes for expressing the biosensor ofclaim 1 in a red blood cell, the combination comprising a firstexpression cassette and a second expression cassette, wherein the firstexpression cassette encodes the first protein and a second expressioncassette encodes the second protein.
 13. A biosensor comprising: (a) afirst protein comprising: (i) a first extracellular ligand bindingdomain, (ii) a first transmembrane domain, (iii) an extracellular linkercomprising 7-25 amino acids and connecting the first extracellularligand binding domain and the first transmembrane domain, (iv) anintracellular first fragment of a functional protein, and (v) anintracellular linker comprising 7-25 amino acids and connecting thefirst transmembrane domain and the intracellular first fragment of thefunctional protein; and (b) a second protein comprising: (i) a secondextracellular ligand binding domain, (ii) a second transmembrane domain,(iii) an extracellular linker comprising 7-25 amino acids and connectingthe second extracellular ligand binding domain and the secondtransmembrane domain, (iv) an intracellular second fragment of afunctional protein, and (v) an intracellular linker comprising 7-25amino acids and connecting the second transmembrane domain and theintracellular second fragment of the functional protein; wherein thefirst extracellular ligand binding domain and the second extracellularligand binding domain bind to the same ligand, wherein the firstfragment of the functional protein and the second fragment of thefunctional protein are capable of interacting to reconstitute activityof the functional protein, and wherein linkers comprise a sequence ofamino acids selected from glycine, serine, and combinations thereof. 14.The biosensor of claim 13, wherein the intracellular linkers eachcomprise the sequence GGGSGGGS.
 15. The biosensor of claim 13, whereinthe functional protein is a fluorescent protein and the fluorescentprotein emits fluorescence when the ternary complex is formed and thefirst fragment of the fluorescent protein and the second fragment of thefluorescent protein interact to reconstitute the fluorescent protein.16. The biosensor of claim 15, wherein the fluorescent protein isselected from the group consisting of green fluorescent protein (GFP),EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP,AcGFP, ZsGreen, T-Sapphire, GFP-S65T, frGFP, sfGFP, EBFP, EBFP2,Azurite, mTagBFP, ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1,Midori-Ishi Cyan, TagCFP, mTFP1 (Teal), Dronpa, EYFP, Topaz, Venus,mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, Kusabira Orange,Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP,TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed- Monomer, mTangerine,mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, mKate, HcRed1,mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, and AQ143.
 17. Thebiosensor of claim 13, wherein the functional protein is an enzyme andthe enzyme exhibits enzymatic activity when the ternary complex isformed and the first fragment of the enzyme and the second fragment ofthe enzyme interact to reconstitute the enzyme.
 18. The biosensor ofclaim 17, wherein the enzyme is a luciferase.
 19. A combination ofexpression cassettes for expressing the biosensor of claim 13 in a redblood cell, the combination comprising a first expression cassette and asecond expression cassette, wherein the first expression cassetteencodes the first protein and a second expression cassette encodes thesecond protein.